發(fā)布時(shí)間：2018-06-18 03:38

  本文選題：乳桿菌 + 耐藥性　； 參考：《河北農(nóng)業(yè)大學(xué)》2012年碩士論文

【摘要】：乳桿菌在自然界分布廣泛,且作為益生菌廣泛應(yīng)用于食品、飼料和醫(yī)藥衛(wèi)生等相關(guān)領(lǐng)域。近年來,越來越多的研究表明,抗生素的廣泛應(yīng)用在治療疾病的同時(shí),也使得環(huán)境中耐藥菌株的種類及數(shù)目增加,攜帶有可轉(zhuǎn)移耐藥因子的益生菌應(yīng)用于生產(chǎn),將嚴(yán)重影響到食品的安全性。本文對(duì)從食品環(huán)境中分離保存的34株乳桿菌的抗生素敏感性進(jìn)行了研究,首先從具有較高耐藥性菌株中檢測(cè)是否存在質(zhì)粒DNA,探討了在胃腸條件下含質(zhì)粒菌株生長的耐受性;采用質(zhì)粒消除法分析了菌株質(zhì)粒譜與耐藥譜之間的聯(lián)系,探索了乳桿菌耐抗生素的分子機(jī)制,旨在為益生乳桿菌安全性評(píng)價(jià)體系建立提供理論依據(jù)。主要結(jié)果如下: 1、抗生素敏感性測(cè)定結(jié)果表明,供試乳桿菌菌株對(duì)桿菌肽、多粘菌素B、卡那霉素以及萘啶酸的耐藥率達(dá)到了100%;對(duì)萬古霉素的耐藥率為82.4%(28/34);對(duì)鏈霉素的耐藥率為91.2%(31/34);對(duì)慶大霉素的耐藥率為88.2%(30/34),表明供試菌株對(duì)這幾種抗生素具有普遍的耐藥性,耐藥譜的分布表現(xiàn)出種間差異性。供試菌株對(duì)青霉素的耐藥率為38.2%(13/34);對(duì)頭孢噻吩的耐藥率為17.6%(6/34);對(duì)氨芐西林的耐藥率為41.2%(14/34);對(duì)四環(huán)素和氯霉素的耐藥率為8.8%(3/34);對(duì)利福平的耐藥率是29.4%(10/34),表明供試菌株對(duì)這幾種抗生素相對(duì)比較敏感,且敏感性分布具有種屬特異性。 2、選擇耐藥性相對(duì)較強(qiáng)的16株菌提取質(zhì)粒,最終確定有6株含有質(zhì)粒,其中菌株CH8、Lp-A和Lp-4為戊糖乳桿菌,LLB為瑞士乳桿菌,LL為嗜酸乳桿菌,L11為植物乳桿菌。模擬胃腸環(huán)境生存實(shí)驗(yàn)表明,含質(zhì)粒菌株CH8、Lp-A、LLB、LL和L11能夠在胃腸環(huán)境下生存下來,這意味著質(zhì)粒上攜帶抗性基因的菌株或許會(huì)對(duì)寄主健康帶來潛在危害。 3、利用質(zhì)粒消除法證實(shí)菌株CH8質(zhì)粒上可能含有氯霉素和β-內(nèi)酰胺類的抗性基因。PCR分析證實(shí)菌株CH8上擁有大小為10kb左右的兩條質(zhì)粒,測(cè)序表明兩條質(zhì)粒上含有β-內(nèi)酰胺類的抗性基因blr。進(jìn)一步地,將來自菌株CH8的耐藥質(zhì)粒轉(zhuǎn)化于對(duì)氯霉素敏感的大腸桿菌HB101中,觀察到大腸桿菌HB101在含有氯霉素的培養(yǎng)基上能夠生長。 綜上,在敏感性及耐藥性質(zhì)粒檢測(cè)基礎(chǔ)上,評(píng)價(jià)含耐藥質(zhì)粒菌株在胃腸環(huán)境的生存能力,將在腸胃逆境下生存菌株進(jìn)行質(zhì)粒消除,以此探討質(zhì)粒譜與耐藥譜之間的聯(lián)系,再將菌株質(zhì)粒轉(zhuǎn)化到抗生素敏感的大腸桿菌細(xì)胞中,可以形成一條新的潛在益生乳桿菌抗生素敏感性的安全性評(píng)價(jià)程序。
[Abstract]:Lactobacillus is widely distributed in nature and is widely used as probiotics in food, feed, medicine and hygiene. In recent years, more and more studies have shown that the widespread use of antibiotics in the treatment of diseases, but also to increase the variety and number of drug-resistant strains in the environment, carrying transferable resistance factors of probiotics used in production, Will seriously affect the safety of food. In this paper, the antibiotic sensitivity of 34 strains of Lactobacillus isolated from food environment was studied. The relationship between plasmid profile and drug resistance spectrum of Lactobacillus was analyzed by plasmid elimination method, and the molecular mechanism of antibiotic resistance of Lactobacillus was explored in order to provide theoretical basis for the establishment of safety evaluation system of probiotic Lactobacillus. The main results are as follows: 1. The results of antibiotic sensitivity test showed that Lactobacillus strains were resistant to bacilli peptide. The resistance rates of polymyxin B, kanamycin and naphthalic acid reached 100; vancomycin resistance rate was 82.4- 28 / 34%; streptomycin resistance rate was 91.21% / 34%; and gentamicin resistance rate was 88.2% 30% 34%, indicating that the strains tested had universal resistance to these antibiotics. The distribution of drug resistance spectrum showed interspecific differences. The resistance rate of the tested strains to penicillin was 38.2% / 34%; to cefthiophene was 17.6 / 634%; to ampicillin was 41.22% / 34%; to tetracycline and chloramphenicol was 8.88% / 34%; and to rifampicin was 29.44% to rifampicin, indicating that the strains were resistant to these strains. Antibiotics are relatively sensitive. The sensitivity distribution was species-specific. 2. 16 strains with relatively strong drug resistance were selected to extract plasmids, and 6 strains were found to contain plasmids. Among them, strains CH8 Lp-A and Lp-4 were Lactobacillus pentanaccharis, LLB, Lactobacillus Switzerland, LLL, Lactobacillus acidophilus, Lactobacillus acidophilus, L11, Lactobacillus plantarum. The experimental results of simulated gastrointestinal survival showed that plasmid strains CH8G Lp-An LLBNLL and L11 could survive in gastrointestinal environment. This means that the strain carrying the resistance gene on the plasmid may be a potential hazard to the host health. 3. The plasmid elimination method was used to confirm that the plasmid of strain CH8 might contain chloramphenicol and 尾-lactam resistance genes. PCR It was confirmed that the strain CH8 had two plasmids about the size of 10kb. Sequencing showed that the two plasmids contained 尾 -lactam resistance gene blr. Furthermore, the resistant plasmid from strain CH8 was transformed into Escherichia coli HB101, which was sensitive to chloramphenicol. It was observed that E. coli HB101 could grow on the medium containing chloramphenicol. On the basis of sensitivity and drug resistance plasmid detection, the survivability of drug-resistant plasmid strains in gastrointestinal environment was evaluated, and the plasmid elimination was carried out under gastrointestinal stress, so as to explore the relationship between plasmid profile and drug resistance spectrum. A new procedure for evaluating the antibiotic sensitivity of Lactobacillus probiotics was established by transforming the plasmid into E. coli cells sensitive to antibiotics.
【學(xué)位授予單位】：河北農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R378

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